4-Aminoimidazole[4-¹⁴C]-5-carboxamide hydrochloride (AICA-¹⁴C) was synthesized in order to use it as the radioactive biological tracer to follow the biosynthetic mechanism of polynucleotide purines and the behavior of antimetabolites expected to act during the course of such biosysnthesis. The synthetic route started with barium carbonate-¹⁴C and went through 7 steps including sodium cyanide-¹⁴C, malon-amamidine[3-¹⁴C]hydrochloride, and formamidomalonamamidine[3-¹⁴C]hydrochloride hemihydrate, with a final yield of 23% of AICA-¹⁴C.